The samples were 91912014 1H13-16 9/10/11/12.
First the kit assumes that the samples are stored or processed with liquid nitrogen as it asks you to used a mortar and pestle to grind the tissue into powder. Our samples are not preserved this way so I modified the first step to work with our samples.
Before beginning the procedure I also mixed all the reagents necessary for the kit. I added Absolute Ethanol to the DNA wash and pure Isopropyl alcohol to the HBC buffer in the quantities required. These bottles have been labelled accordingly for future reference.
The protocol is as follows:
- Added 350 ul ML1 Buffer
- Added 25 ul Proteinase K solution
- Used pestle in homogenization tube to grind tissue in solution
- Vortexed
- Incubated at 60 C for 30 minutes
- Added 350 ul Phenol:Chloroform:Isoamyl Alcohol (25:24:1)
- Vortexed
- Centrifuged 10,000 g for 2 minutes
- Transferred the upper aqueous phase to new tube (~300 ul)
- Added 300 ul MBL Buffer
- Added 10 ul RNase A
- Vortexed for 15 seconds
- Incubated at 70C (started at 67.5 C) for 10 minutes
- Cooled to room temperature sitting for 5 minutes
- Added 300 ul 100% EtOH
- Vortexed for 15 seconds
- Put spin column in collection tube
- Added 750 ul sample solution to column
- Centrifuged at 10,000 g for 1 minute
- Discarded flowthrough
- Repeated 18-20 with remaining sample
- Discarded collection tube and replaced with a new one.
- Added 500 ul HBC solution.
- Centrifuged at 10,000 g for 30 seconds
- Discarded flowthrough
- Added 700 ul DNA Wash Buffer
- Centrifuged at 10,000 g for 1 minute
- Discarded flowthrough
- Repeated 26-28
- Centrifuged Empty column for 2 minutes at 10,000 g
- Discarded collection tube and put column into microcentrifuge tube for sample collection
- Added 100 ul preheated 70C Elution Buffer
- Incubated for 2 minutes
- Centrifuged at 10,000 g for 1 minute
- Repeated 32-34.
- Stored DNA at -20 C
Tomorrow I will run the DNA out on a gel to check for quality.
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